Trypsin

TRYPsin, an enzyme found in pancreatic juice, was described by Kiihne in 1867 and belongs to the group of proteases. It breaks down proteins, protamines, peptones, and polypeptides. In its purest form, free from lipase and amylase, it was obtained by the method of Willstätter: after dehydration and defatting the gland, the enzymes are extracted with glycerin and lipase and amylase are removed by adsorption on kieselguhr; in the solution remains T. with a small amount of amylase; further purification of T. is achieved by adsorption on kaolin. It has also been obtained in the form of protein crystals (Northrop), which do not guarantee either its purity or its chemical individuality. Data on the optimal temperature of action of T. are extremely contradictory (from 38° to 60°), which is apparently explained by the presence of various impurities. The same applies to data on its stability. After combination with enterokinase, the stability of T. is significantly reduced. At a weakly acidic reaction (pH about 5), the stability of T. is greatest; it slowly decreases with a change in reaction to neutral and then falls rapidly with an increase in the concentration of OH'. The presence of proteins and especially the products of their digestion makes T. more resistant to heating, so that trypsin mixed with a solution of peptone can even withstand boiling, which can be used to neutralize trypsin solutions. Trypsin acts best at a weakly alkaline reaction corresponding to a 0.3–0.4% solution of soda; the action of alkalis is most favorable at a concentration of hydroxyl ions equal to n/70–n/100 of the solution; T. acts more weakly at a neutral and weakly acidic reaction; the reaction optimum depends strongly on the substrate (see Digestion); on average it lies at pH = from 8.0 to 8.7. Under the influence of acids, T. quickly loses activity: at pH = 10^-4 it does not act; a 0.1% solution of hydrochloric acid destroys its action, but bound HCl, small amounts of organic acids do little harm to the action of T. The accumulation of products of digestion reaction slows down the rate of splitting; a slowing action is exerted by a 10% solution of NaCl, sodium sulfate, salts of heavy metals, as well as adsorbents—animal charcoal, talc, kaolin, serum albumin, serum. The inhibitory action on T. is exerted by antitrypsin, a substance which, in the opinion of some authors, prevents the autodigestion of the intestinal wall and appears in blood plasma during various cachexias as a result of the appearance in the blood of enzymes of autolysis. Antitrypsin withstands brief boiling. Thymol, chloroform, toluene, sodium fluoride also slightly delay the action of T. Pepsin in the presence of HCl digests T. Accelerating action is exerted by various salts, especially Ca and Mg, a very weak solution of NaCl (0.05%), the presence of borax, a negligible amount of potassium cyanide, liver juice, as well as substances formed from pancreatic gland tissue when lying in the air or when alcohol is added. T. from pure fistula pancreatic juice acts only on peptones, polypeptides, and protamines; on unchanged proteins it acts only in the presence of enterokinase (see Digestion), discovered by Pavlov and Shapovalnikov in intestinal juice and, in their opinion, produced by the mucous membrane of the small intestines. Waldschmidt-Leitz considers that enterokinase in the form of pro-kinase is secreted together with T. by the cells of the pancreas and activated in the small intestines by erepsin. This question, however, is not sufficiently elucidated; according to other authors, the action of kinase in the cells of the pancreas is paralyzed by special substances produced in them. In connection with the diverse action of T., various authors propose using different terms to denote it, emphasizing one or another of its actions, for example, trypsinogen, protrypsin, an inactive protein T. and activated by kinase; Waldschmidt-Leitz introduced the term trypsin-kinase, i.e., trypsinogen activated by kinase; Oppenheimer proposes to distinguish trypsinase, a pure protease, and polypeptidase, etc. The action of T. (trypsin-kinase) on various proteins is not the same: raw fibrin is most easily subject to its action; cooked fibrin and chicken protein act more slowly; serum globulin and serum as a whole are split only after preliminary action of gastric juice; T. also splits gluten, collagen after action on them of gastric juice; it cleaves nucleic acids from nucleoproteids, digests nucleoalbumins, mucus, the membrane of fat cells and parenchymatous organs; oxyhemoglobin is split into heme and protein body; T. of higher animals does not digest chitin and keratin. In relation to its action on polypeptides, T. apparently belongs to carboxypolypeptidases, since its action is connected with the presence of a free carboxyl group; in this case, the amino acid to which this group belongs is significant. In general, the action of T. depends on the presence of certain amino acids and on the order of their arrangement in the polypeptide chain, and not on the length of the chain, as was thought earlier. For example, polypeptides containing tyrosine and tryptophan are split by T. most easily; the presence of leucine, alanine, aspartic and glutamic acids slows down its action; proline, hydroxyproline, and glycocol are not split at all; the splitting of artificial polypeptides occurs asymmetrically. T. is present in all animals having a pancreas; in the embryo it appears in the last third of intrauterine life; in birds and cold-blooded animals T. is studied little. The secretion of T., as established by I. P. Pavlov, Bayliss, Starling, and others, is regulated not only by the nervous system but also by a hormonal path (see Digestion, Secretin). The fate of T. in the intestine is not elucidated; its digestion by pepsin and HCl is protected by bile, which precipitates pepsin on the villi. The T. contained in the feces can only be partly attributed to T. of pancreatic juice, since it can arise from leukocytes and bacteria. To detect T., the serum plate method of Loeffler is used: a Petri dish is poured with horse, bull, or camel serum to a layer of 1/2 cm and heated at 70° until coagulation; serum is dropped with the test solution of the enzyme and left to stand for 24 hours at 50°; the appearance of pits shows the presence of T. Serum plates can be replaced by plates from Milch-agar, synthetic polypeptides of Abderhalden, or peptone of Wite, La Roche, etc. For quantitative determination of T., there are many methods, the basis of which is the determination of the amount of protein before the action of the enzyme and after splitting. The most suitable is the method of Willstätter and Persiel, since it takes into account the degree of activation and the use of large amounts of substrate with a small amount of trypsin. Required reagents: 1) 15% solution of gelatin; 2) solution of T.: 1–2 g of pancreas is rubbed with a small amount of water, the resulting mass is diluted with 100 cm³ of water, first slowly, then faster and immediately filtered. It is better to use the first portions of the filtrate; 3) extract of enterokinase: 5 g of dried mucous membrane of small intestines is infused for 2 hours with 250 cm³ of n/2 solution of NH₃ at 30°; the resulting extract is evaporated in a Faust-Heim apparatus; 4) buffer solution: nNH₃ : NH₄Cl in a ratio of 1:2; 5) solutions for titration by the Willstätter-Waldschmidt-Leitz method. One unit is taken as the amount of enzyme giving an increase in acidity corresponding to 2 cm³ of n/100 KOH. To determine 0.01–0.25 g of dried gland or pure preparation obtained from it, it is mixed in test tubes with ground stoppers with 0.3 cm³ of solution of enterokinase and brought up to 1.5 cm³ with water and then heated for 1/2 hour at 37°. After bringing the volume to 5 cm³ with water, 2 cm³ of n-solution of NH₃+NH₄Cl are added, then 5 cm³ of a 15% solution of gelatin, heated in a thermostat, is quickly added from a heated pipette and well mixed. After 20 min. the contents of the test tube are poured into 55 cm³ of boiling absolute alcohol, the test tube is rinsed with the same amount of alcohol, brought to an alkaline reaction with thymolphthalein by adding n/5 KOH and titrated. A number of other methods are suitable for the determination of T.: Grutzner, Volhard-Lohlein, etc. T. does not follow the rule of Schütz-Borisov: the amount of digested protein is directly proportional to the amount of enzyme and the time of its action, m. karagana.